Methamphetamine (MA) addiction is a pervasive public health concern in the U.S. and is associated with violent crime, mental health decline, and a high rate of relapse in recovering patients. Despite decades of research, there are still no FDA-approved treatments for MA addiction. Both genetic and environmental factors contribute to the etiology of MA addiction; however, genome-wide association studies in humans are limited in their power to identify common variants associated with this devastating disease. Mouse genetics offers a powerful, complementary tool for accelerating novel gene discovery and biological mechanisms. We recently identified a 210 kb region on mouse chromosome 11 containing two protein coding genes (Hnrnph1 and Rufy1) that contributes to the locomotor stimulant response to MA. The primary objective of this proposal is to identify the novel quantitative trait gene (QTG) and the neurobiological mechanism by which this QTG regulates the stimulant and addictive properties of MA. In Aim 1, we will identify the QTG(s) that regulates MA sensitivity. We generated a 112 kb congenic mouse containing only Hnrnph1 and Rufy1, providing the unique opportunity to determine whether this region is both necessary and sufficient for MA sensitivity. Furthermore, we recently used transcriptional activator-like effecto nucleases (TALENs) to introduce null mutations in Hnrnph1 and Rufy1. Preliminary studies provide strong, replicable evidence that Hnrnph1 is a QTG for reduced MA sensitivity. To fully tackle all of the polymorphisms within the 210 kb region that could potentially contribute to behavior, we will also delete a 150 kb intergenic region using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to introduce double stranded breaks that flank the intergenic region. Finally, we will use CRISPR/Cas9 to generate knockin mice carrying SNPs from the candidate QTG. In Aim 2, we will use transcriptome analysis of the striatum and ventral midbrain via mRNA sequencing (RNA- seq) in mice carrying the congenic allele, the knockout allele of the QTG, or both alleles relative to wild-type mice to hone the molecular pathways and neurobiological mechanisms that link genetic variation with behavior. Preliminary analysis of a congenic line capturing the QTL for reduced MA sensitivity suggests perturbations in midbrain dopaminergic neuron development, glutamate signaling, and adrenergic signaling as potentially responsible for a reduction in MA-induced behavior. In Aim 3, we will use in vivo microdialysis, immunoblotting, immunohistochemistry, and operant oral MA self-administration in congenics and knockouts to functionally assess the neurochemical mechanism by which the QTG influences the stimulant and addictive properties of MA. The proposed studies employ a unique combination of genetics, genomics, and functional analyses to uncover the neurobiological mechanism by which a novel genetic factor contributes to addiction-relevant behaviors. These findings will likely have translational relevance in the genetic and molecular mechanisms that confer susceptibility/resilience to MA addiction and could inform new therapeutic avenues.